Partial purification and properties of respiratory chain-linked l-glycerol 3-phosphate dehydrogenase from a marine bacterium, Vibrio alginolyticus.
Respiratory chain-linked L-glycerol 3-phosphate (G3P) dehydrogenase [EC 1.1.99.5] of marine bacterium, Vibrio alginolyticus, was extracted from the membrane fraction by treatment with Tween 20, and fractionation on DEAE-Sephacel and QAE-Sephadex in the presence of 0.05% Liponox DCH(alkyl polyoxyethylene ether) yielded a preparation having a specific activity of 22.1 units/mg protein when assayed by phenazine methosulfate (PMS)-coupled reduction of thiazolyl blue tetrazolium (MTT). The purified enzyme had an apparent molecular weight of 300,000 as determined by chromatography on Sepharcyl S-300 in 0.05% Liponox DCH, and had noncovalently bound FAD as its coenzyme. The enzyme had a pH optimum of 8.5-9.0, and required 200 mM NaCl or KCl and an appropriate detergent (such as Tween, Brij or Liponox DCH) for maximum activation. The activating effect of NaCl was due to a decrease in Km for G3P and that of Tween 20 was due to both a decrease in Km and an increase in Vm. Triton X-100 could not activate the enzyme and was inhibitory in the presence of phospholipids. The reaction followed a ping-pong mechanism. IN addition to PMS, 2,6-dichlorophenol indophenol and duroquinone, the enzyme could reduce ubiquinone-5 (Q-5) in the presence of Liponox DCH at a rate of 46% of the PMS reductase activity. The enzyme was strongly inhibited by heavy metal ions and by p-chloromercuribenzoate. The activity for Q-5, but not for PMS, was inhibited by o-phenanthroline and bathophenanthroline, suggesting the participation of nonheme iron protein in the Q-5 reduction.
D002462:Cell Membrane / Q000378:metabolism; D002712:Chlorides / Q000494:pharmacology; D003580:Cytochromes / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D005990:Glycerol / Q000378:metabolism; D005993:Glycerolphosphate Dehydrogenase / Q000302:isolation & purification*; D010743:Phospholipids / Q000494:pharmacology; D011190:Potassium Cyanide / Q000494:pharmacology; D011809:Quinones / Q000378:metabolism; D012995:Solubility; D014733:Vibrio / Q000201:enzymology*
D002712:Chlorides; D003580:Cytochromes; D003902:Detergents; D010743:Phospholipids; D011809:Quinones; D005993:Glycerolphosphate Dehydrogenase; D011190:Potassium Cyanide; D005990:Glycerol